Herpes simplex virus (HSV) contains a double-stranded linear DNA genome comprised of approximately 152 kb of nucleotide sequence, which encodes about 80 genes. The viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in the transcription and subsequent synthesis of gene products in roughly discernible phases: Immediate Early (IE, or .alpha.), Early (E, or .beta.) and Late (L, or .gamma.). Immediately following the arrival of an HSV genome into the nucleus of an infected cell, the IE genes are transcribed. The IE genes are all activated by a complex including the HSV virion particle VP16 and the cellular factor, Oct-1, which binds to a consensus sequence (TAATGARAT) regulating IE gene expression (Preston et al., Cell, 52, 425-35 (1988)). The presence of this sequence, thus, confers the IE quality to HSV regulatory sequences. The efficient expression of IE genes, thus, does not require prior viral protein synthesis, while later expression depends upon the presence of IE gene products. The products of IE genes are required to activate transcription and regulate the remainder of the viral genome.
Infected Cell Peptide 4 (ICP4), ICP0, ICP27, ICP22, and ICP47 are the immediate early gene products, and these show varying degrees of essentiality to HSV function. These phosphoproteins possess regulatory activities thought to prime the host cell for the efficient cascade of subsequent viral gene expression, DNA replication and the production of progeny virions. The manner in which the IE gene products act in concert to effect the cascade of regulatory activity giving rise to productive infection is poorly understood. However, many functions of these gene products are known. For example, ICP0 activates most test promoters in transient assays (Quinlan and Knipe, Mol. Cell. BioL, 5, 957-63 (1985)), elevates levels of viral gene expression and growth in tissue culture and in the trigeminal ganglia (Cai and Schaffer, J Virol., 66, 2904-15 (1992)), and facilitates the reactivation of virus from latency (Leib, et al., J Virol., 63, 759-68 (1989)). ICP27 modulates the activity of ICP4 and ICP0, and it regulates viral and cellular mRNA processing. These activities of ICP27 mostly contribute to efficient DNA replication, hence it is essential for viral growth: however. ICP27 also regulates the proper expression of early and late genes. ICP22 promotes efficient late gene expression in a cell-type dependent manner and is involved in the production of a novel modified form of RNA Pol II. ICP4 is a large multifunctional protein. It can act as a transcription factor that either represses or activates transcription through contacts with the general transcriptional machinery. ICP4 is absolutely required for both virus infectivity and the transition from IE to later transcription.
The activities of genes other than the IE genes also play significant regulatory roles in HSV infection. The HSV gene UL39 encodes ICP6, the large subunit of ribonucleotide reductase, a key enzyme in the pathway reducing ribonucleotides to the corresponding deoxribonucleotides. ICP6 (or UL39) is best classified as an early gene and is not essential for viral growth in dividing cells. UL41 is a late HSV gene product released during host cell infection which mediates the inhibition of host cell metabolism, including DNA and protein synthesis. This viral induced host shut-off is linked to destabilization and degradation of host mRNA.
Owing to its central role in the regulation of HSV gene expression, ICP4 has been the subject of numerous genetic and biochemical studies aimed at developing mutant viruses devoid of ICP4 activity, such as the d120 HSV virus (DeLuca, et al., J Virol., 56, 558-70 (1985); DeLuca, et al., Nuc. Acids Res., 15. 4491-11 (1987); DeLuca, et al., J Virol., 62, 732-43 (1988); Paterson, et al., Virology, 166, 186-96 (1988); Paterson, et al., Nuc. Acids Res., 16, 11005-25 (1988); Shepard, et al., J Virol., 63, 3714-28 (1989); Imbalzano, et al., J Virol., 65, 565-74 (1991); and Shepard, et al., J Virol., 65, 787-95 (1991)). One property of viruses deleted for ICP4 that makes them desirable for gene transfer is that they only express a very limited subset of HSV genes including the four other IE genes: ICP0, ICP27, ICP22 and ICP47, as well as ICP6 (DeLuca, et al., 1985, supra). This set of genes excludes genes encoding proteins that direct viral DNA synthesis, as well as the structural proteins of the virus, many of which interfere with host cell metabolism.
The phenotype of viruses lacking ICP4, suggests that such viruses could be potentially useful for gene transfer purposes (Breakefield, et al., Treatment of Genetic Diseases, (Churchill Livingstone, Inc., 1991); and Chocca, et al., The New Biologist, 2, 739-46 (1990)). Despite the fact viruses lacking ICP4 are blocked at the earliest stage of infection genetically possible subsequent to the delivery of the genome to the host cell nucleus, two phenomena have complicated the use of such viruses for effective gene transfer or therapy. First, viruses lacking essential genes, such as ICP4- or ICP27-deficient viruses, require the exogenous supply of the missing viral gene product, such as a cell line engineered to express the gene (DeLuca, 1985, supra). Homologous recombination events between the mutant viral genome and the wild-type gene resident in the host cell genome can "rescue" a population of viruses no longer deleted for that gene (Id.), particularly where the viral and host cell genomes include sequences of homology. Secondly, despite only expressing the four other immediate early proteins, ICP4-deficient viruses are toxic to infected cells. For example, infection of cells with ICP4 mutants causes chromosomal aberrations and rapid cell death (Johnson, et al., J Virol., 66, 2952-65 (1992); Peat and Stanley, J Gen. Virol., 67, 2273-77 (1986)). Moreover, either ICP4, ICP0, ICP27 or ICP22 significantly reduce the transformation efficiency of cultured cells via G418 resistance (Johnson, et al., J Virol., 68, 6347-62 (1994)). This toxicity is most probably due to the expression of one or more of the remaining immediate early proteins, rather than the incoming capsid, since defective HSV virus particles, containing intact capsids and lacking all IE genes, are not toxic. In addition, ICP4 deficient viruses shut off host cell protein synthesis through the activity of the UL41 virion gene product (Read, et al., J Virol., 67, 7149-60 (1993)).
Further work has produced an HSV vector (d92) having mutations in both essential immediate early genes (i.e., ICP4 and ICP27) using a novel cell line (26 cells) expressing both viral proteins (Samaniego et al., J Virol. 69(9), 5705-15 (1996)). This viral/cell line system was engineered to minimize homology between the virus and the cell line in order to substantially reduce the probability of rescuing a wild-type revertant HSV, thus permitting the use of higher m.o.i. in gene-transfer protocols. Moreover, more persistent genomes are obtainable with d92 than with ICP4.sup.(-) viruses. In the absence of ICP4 and ICP27, only ICP6, ICP0, ICP22, and ICP47 are expressed from the viral genome; hence the double mutant is somewhat less cytotoxic than the single mutant. However, the expression of this set of genes renders the double mutant significantly cytotoxic, largely due to the presence of the ICP0 gene product. Despite the teachings in the art indicating that ICP0.sup.(-) viruses can grow in the absence of complementation (e.g., Cai and Schaffer, supra), the isolation and propagation of an HSV wherein ICP0 is deleted from an HSV genome already lacking ICP4 and ICP27 by employing a double complementing (E26 cells) cell line has not been possible. The growth-dampening effect of deleting ICP0 in a ICP4.sup.(-) ICP271.sup.(-) mutant is unexpectedly greater than that observed for a wild-type background.
Therefore, a need exists for defective herpes simplex virus strains exhibiting efficient growth in a controlled laboratory complementing system, a reduced level of wild-type virus regeneration, and lowered cytotoxic effects. Concomitantly, there exists a need for a method of producing such viral strains and a cell line for propagating the same.